Oral Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Proteins involved in the adherence of Mycoplasma hyopneumoniae to abiotic surfaces and porcine monolayers which may also play a role in biofilm formation (#15)

Benjamin Raymond 1 , Manfred Rohde 2 , Matthew Padula 1 3 , Gareth Maglennon 4 , Andrew Rycroft 4 , Steven Djordjevic 1
  1. The ithree Intitute, University of Technology, Sydney, NSW, Australia
  2. Helmholtz Centre for Infection Research, University of Braunschweig, Braunschweig, Germany
  3. Proteomics Core Facility, University of Technology, Sydney , NSW, Australia
  4. Royal Veterinary College, University of London, London, United Kingdom

Mycoplasma hyopneumoniae is a genome-reduced pathogen which colonises the porcine respiratory tract causing a chronic pneumonia. Consequently this, in addition to the host immune response leads to reduced feed conversion within swine herds and a significant financial burden inflicted upon the industry. In order to develop successful vaccines a fundamental understanding of M. hyopneumoniae virulence mechanisms is required. Biofilm formation is one of the ways in which a number of chronic respiratory pathogens persist within their host. Here we have used time-lapse microscopy to monitor biofilm formation of M. hyopneumoniae on abiotic surfaces. M. hyopneumoniae formed prolific biofilms after prolonged incubation on a glass surface and was accelerated when cultured on a porcine epithelial cell monolayer (PK-15). In order to study the adherence of M. hyopneumoniae to these monolayers, we devised a global methodology to identify those proteins which are involved in this process. Surface proteins from PK-15 monolayers were biotinylated and bound to an avidin column. This column containing the labelled surface proteins was incubated with a native M. hyopneumoniae lysate. After extensive washing, M. hyopneumoniae proteins which bound to the column were eluted in 2M NaCl and additionally in 0.4% Trifluoroacetic acid to remove any strongly bound proteins. These proteins were separated by 1D-SDS-PAGE, in-gel trypsin digested and analysed by LC-MS/MS. 75% of the proteins identified have been found by our lab to reside on the M. hyopneumoniae cell surface and are deemed putative adhesins. One of these adhesins, MhpX was found to be important for biofilm formation. Transposon mutants in MhpX appear to have a profound effect on biofilm formation. We have also identified a number of additional putative biofilm associated genes from our high-throughput biofilm screens. Further work will be needed to characterise these proteins and examine their role in the pathogenesis of M. hyopneumoniae.